In the past, research on aspartate transcarbamoylase (ATCase) of E. coli has been focused primarily on clarifying the nature of allosteric transition whereby the enzyme is converted from a low- affinity T-state to a high-activity R-conformation, thereby contributing to the regulation of pyrimidine biosynthesis. With the application of site-directed mutagenesis to produce altered catalytic and regulatory chains coupled with recent knowledge of the structure of ATCase from crystallography, it is possible to extend these studies to mutant forms of ATCase having weakened subunit interactions and to hybrids containing different numbers of active sites. The uncertainties in analyzing enzyme kinetics resulting from the inability to differentiate between substrate binding and catalytic turnover will be circumvented by direct equilibrium dialysis measurements of the binding of a radioactively-labeled bisubstrate analog. In this way the thermodynamics of ligation to intact enzyme and to isolated catalytic subunit will be linked to the energetics of assembly of ATCase from subunits for more rigorous tests of potential allosteric mechanisms. Also, the rate of the T leads to R transition will be measured by stepped-flow techniques using ATCase containing spectral probes on the regulatory chains sensitive to the global conformational change. Mutant forms of catalytic trimers serve as model oligomeric proteins for studies of protein stability, folding of the polypeptide chains into domains and communication between the chains. Interchain and intrachain interactions are being probed by a hybridization technique coupled with differential scanning microcalorimetry and denaturation by guanidine HC1. With either tyrosine or histidine substituted at selected positions, 1H-NMR and 13C-NMR will be used to study the independent effects of substrate and analogs on the motion of flexible loops in the catalytic trimers. Incomplete polypeptide chains produced by deletion mutations in pyrB which encodes the catalytic chains of ATCase associate in vivo to produce active trimers which serve as in vitro models for studying folding of chains and their assembly to form active enzyme. This system can be used to test the chain-displacement mechanism of interallelic complementation. Mutants which are inactive because of incorrect folding of the chains ar being used in a search for second-site revertants and for intergenic suppression by mutations in pyrl which encodes the regulatory chains. Potential transition-state analogs conversion of substrate to product are being synthesized for studies of the catalytic mechanism and to identify essential amino acid residues. Also, measurement of isotope effects on mutants of low activity and NMR studies aimed at identifying possible proton donors will be used to test proposed catalytic mechanisms. Through detailed understanding of the allosteric transition and catalytic mechanism of ATCase, knowledge of pyrimidine and nucleic acid metabolism will be greatly enhanced.